Process of purifying dehydrocholic acid



Patented Dec. 8, 1953 Paocass or PURIFYING harmonio- .oHoLIo ACID Pierre Poulenc, Paris, Bernard Gauthier, Antony, .and Georges Marie Emile .Aloys Kirchhoffer, Paris, France, assignors :to Societe: Chimie 'et Atomistique, Paris, France, a .French body corparate Application March :29, 1952, Serial Nub-79,438

Claims priority, application France -April.,20,1v951 (Cl. 2GB-3971) 9 Claims.

The presen-t invention .relates to `a process of purifying dehydrocholic acid.

It is known that this acid is obtained by conversion of cholic acid itself extracted from bovine bile which contains normally 3 to 3.5% of choli'c acid or i3, '7, 12 trihydroxy cholanic acid (Fig. I of annexed drawing) apart from cuantities seven to eight times weaker oi desoxyeholc acid, or r3, .12 dihydroxy cholanic acid yand traces of .other related hydroxyl cholanic acids.

.Dehydrocholic acid or 3, '7, l2 lieto cholanic acid (Fig. 2) which is of great therapeutical value if obtained by direct oxydation of three hydroxyl functions of cholic acid into isotonic groupings. To obtain 'this product pure, pure cholc acid is required the preparationV ci which is costly due to the large losses occurring during; purincation, In practice, therefore, it is more advantageous to start with la commercial cliolic acid, but the obtained product is polluted with 3, '12 diketo cholanic .acid coming from the oxydation vor" the principal impurity of chc'lic acid namely desoxycholic acid, and also with exidized biliary pigments that give to the alkaline solutions of dehydrocholic acid an intense yellow or brown colour.

The invention has for its object to remedy these drawbacks by means of a particularly easy and effective process ci purifying dehydrocholic acid.

It has not been discovered that biliary acids possessing a ketonic function at position 3, in an alkaline medium combine with aliphatic halogenated hydrocarbon. I

This vproperty may be likened to the action of chloroform on aldehydes .and ketones repre sented by the formula:

oo onen a Y R' Rf con These compounds are, however stable, `mili-lie compounds of biliary acids with halogenated hydrocarbons which are themselves very miste-ble and due to this regenerate very easily-the starting biliary acid.

This property is particularly interesting in the case of dehydrocholic acid for the combinations of this acid with aliphatic halogenated hydrocarbons, are insoluble in water, as opposed to the analogous combinations of other biliary acids 'having a ketonic function at position 3. The insolubility in water lof the dehydrocholic acid-halogenated hydrocarbon combination permits, then, the easy isolation of dehydrocholic acid Ain a complex mixture of 'biliary acids. lndeed, in this complex mixture, the biliary acids not having a ketonic grouping at position '3, do not react with halogena'ted hydrocarbons and remain dissolved in the water, the biliary acids having a ketonic grouping at position 3 with the exception of dehydrocholi'c `acid yield Awith these hydrocarbons combinations 'solublein water, and alone precipitate the 'dehydrocholic aci-whalegenated hydrocarbon 'combination Awl'iich 'can then be separated.

The dehydrocholic acid may then "be recovered in a high state of purity from this 'combination by decomposing it with excess hot water.

The invention, therefore, has for its object to provi-de a 'process of :purifying dehydrocholic acid, 'which :comprises treating said acid 'the impure state with an :alkaline 'aqueous solution, wherein it is dissolved in the forni of its alkaline salt, thereafter to said .solution at least one aliphatic hal'ogenated hydrocarbcn'tbereby precipitating :an addition compound ofi halogenated hydrocarbon-dehydrocholic acid, there-- after separating this precipitate decomposing it 'with excess hot n'z'ater thereby regenerating 'the alkaline salt of said acid, and lastly liberating fthe dehydrocholic acid 'in the pitre ystate by acidiiication.

When the -dehydrecholic acid to be puriiied includes another biliary acid as an impurity, the combination formed by the rimpurity r' h a halogenated hydrocarbon remains in sold-"on, and this is falso true for the biliary pigments not .aected by this reaction. The process in accordance with the invention permits, therefore. to isolate in the form of an insoluble precipitate the dehydrocholic acid to be puriied.

In yorder to obtain an optimum yield and quality during the purification, certain conditions cf .concentration vmust be fulfilled which depend on the ensuing considerations. In a dilated medium, the yield of the purication 'is insuicient and .in a vmedium too concentratefiv the precipitate formed very voluminous, and it is difiicult to separate from the impurities, and in consearner-ice the depigmentation is Apai-- tici.

' Preferably, therefore, the concentration ci biliary acid in "the aqueous alkaline 'solution is chos-en between 20 and 25%, its solubility being furthermore diminished if necessary by salting with a corresponding alkaline salt such -as ClNa or CIK. v

In this case, the coloured iiltrate obtained after draining of the halogenatedhydrocarbon-dehydrocholic acid combination may still contain less pure dehydrocholic acid. This ltrate may then be subjected to an analogous operation or puriiied by crystallization in alcohol or other solvent according to the purity desired. To collect the precipitate, it may be advantageous to allow it to stand 24 hours in a refrigerator before draining it. The decomposition of the addition compound halogenated hydrocarbon-dehydrocholic acid by water is very easily effected. This is already happening cold with a large amount of excess water, but it is more rapid when the temperature of the water rises, the alkaline salt dissolving in the Water, as it is liberated. It is then very easy to liberate the biliary acid which precipitates by acidification by any convenient acid.

As halogenated aliphatic hydrocarbons which may be used in the process of the invention, may be mentioned among others: chloroform, dichlorethane, carbon tetrachloride, trichlorethylene, ethyl bromide etc.

In order to effect a good precipitation of the addition compound, the halogenated hydrocarbon is used in a proportion at least molecularly equivalent to the treated biliary acid.

In order to render the invention more clear, several non-limitative modes of carrying out the invention will follow.

1n the various exemples, the amounts of the different reactives are expressed in molar equivalents with respect to the amount of dehydrocholic acid employed, the latter being taken each time as unity.

Example 1 60 g1'. (1 part) of commercial dehydrocholic acid (melting point above 215 C.) ere dissolved in a solution of 6 gr. of sodium hydroxide (1 part) in 200 cc. of purified water. The addition of 18 gr. (1 part) of chloroform provokes the immediate formation of the insoluble combination, which is left 24 hours in a refrigerator, drained, and washed with a saturated aqueous solution of sodium chloride.

The precipitate is treated with 900 cc. of puriiied Water. It is slowly dissolved cold, much more rapidly upon heating, liberating chloroform. The obtained solution, substantially uncoloured, ltered, then precipitated by dilute acetic acid, gives after drying, 26 gr. of dehydrocholic acid (melting point 237-238 C.).

The coloured filtrate obtained after draining is precipitated by acetic acid and gives coloured dehydrocholic acid (melting point 210 C.) which is subsequently utilized.

Example 2 80 gr. (1 part) of commercial dehydrocholic acid (melting point above 215 C. )are dissolved in a solution of 11.2 gr. (1 part) of potassium hydroxyde in 250 cc. of purified water. The addition of 30 gr. of 1,2-dichlorethane (1.5 part) provokes the immediate precipitation of the addition combination, which is left 24 hours in a refrigerator, drained, and Washed with an aqueous solution saturated with potassium chloride. The precipitate is treated with 120() cc. of purifled water. It is slowly dissolved cold, then rapidly upon heating, liberating dichlorethane. The rather uncoloured solution obtained is filtered, then precipitated by dilute hydrochloric acid. After drying, 41 gr. of dehydrocholic acid (melting point 23T-238 C.) are recovered.

The coloured ltrate precipitated by the dilute hydrochloric acid yields impurel and coloured de- 4 hydrocholic acid (melting point 215-21'1" C.) Which is utilized in a subsequent operation.

Example 3 60 gr. (1 part) of commercial dehydrocholic acid (melting point 229 C.) are dissolved in a. solution of 6 gr. of sodium hydroxide (1 part) in 200 cc. of purified Water. The addition of gr. of carbon tetrachloride (1.5 part) provokes the immediate formation of the insoluble combination, which is left 24 hours in a refrigerator, drained, and washed with an aqueous solution saturated with sodium chloride. The precipitate is treated with 1000 cc. of purified water. It is slowly dissolved cold, much more rapidly upon heating, liberating carbon tetrachloride. The rather colourless solution obtained is filtered, then precipitated by dilute hydrocholic acid, yields after drying, 33 gr. of dehydrocholic acid (melting ypoint 238 C.)

The coloured filtrate precipitated by dilute acetic acid gives a less pure dehydrocholic acid (melting point 224 C.) which is purified by the same process.

Example 4 gr. (1 part) of commercial dehydrocholic acid (melting point 222 C.) is dissolved in a solution of 5 gr. (1 part) of sodium hydroxide in 200 cc. of distilled water. The addition of 13.4 gr. of trichlorethylene (1 part) provokes the immediate precipitation of the required combination which is left one night in the cold then drained and washed with an aqueous solution saturated with sodium chloride.

The precipitate treated with 900 cc. of distilled Water is slowly dissolved cold, much more rapidly upon heating, liberating trichlorethylene. The rather coloured solution obtained, filtered, then precipitated by dilute acetic acid, yields, after drying, 25 gr. of dehydrocholic acid (melting point 238 C.) The coloured filtrate precipitated by dilute acetic acid gives a yellowish dehydrocholic acid (melting point 214 C.) which is utilized in a subsequent operation or puried by alcoholic crystallization.

Example 5 50 gr. (l part) of commercial dehydrocholic acid (melting point 215o C.) are dissolved in a solution containing 5 gr (1 part) of sodium hydroXide in 200 cc. of purified Water. The addition of 20.5 gr. (1.5 part) of ethyl bromide provokes the immediate precipitation of the required combination which is left one night in the cold, then drained and Washed with a saturated solution of sodium chloride. The precipitate treated with 900 cc. of puriiied Water is dissolved slowly cold, much more rapidly upon heating, liberating ethyl bromide. The rather colourless solution obtained is filtered then precipitated by dilute acetic acid. After dryingy 26 gr. of dehydrocholic acid (melting point 237-238" C.) are recovered.

The coloured filtrate, precipitated by dilute hydrochloric acid yields a yellowish dehydroholic acid (melting point 212 C.) which is puriiied by crystallization in alcohol at C.

Example 6 gr. (1 part) of dehydrocholic acid (melting point 235 C.) are dissolved in an ammonia solution prepared by adding to 400 cc. of puried Water 25 gr. of ammonia at 20%. The solution is thereafter added to ammonium chloride until a light insoluble part appears. The addition of 30 gr. of chloroform (1 part) provokes the immediate precipitation of the required combination. After being left for 1 hour, the crystals are drained, and washed with a concentrated solution of ammonium chloride. The coloured filtrate is conserved. The precipitate treated by 2300 cc. of purified water is dissolved in a water-bath. The obtained solution is filtered then precipitated by dilute acetic acid. After drying, 80 gr. of dehydrocholic acid (melting point 23T-238 C.) are recovered.

There is recovered by precipitation from the coloured filtrate the yellowish dehydrocholic acid, which is puried subsequently by the same process.

Example 7 500 gr. (1 part) of dehydrocholic acid (melting point 229-230D C.) are dissolved in a solution containing 50 gr. (1 part) of sodium hydroxide in 2000 cc. of purified Water. The solution is added to sodium chloride until a precipitate of sodium dehydrocholate (salting) appears.

The addition of 165 gr. (1.1 parts) of chloroform provokes the immediate formation of the insoluble combination, which is drained after being left several hours, washed with a saturated aqueous solution of sodium chloride, then redissolved in 12 litres of hot purified water. The obtained solution is precipitated by dilute acetic acid. About 400 gr. of dry dehydrocholic acid (melting point 235-236 C.) are recovered. The coloured filtrate is added to sodium chloride (which per.

mits a little more of the combination to be recovered), and therefrom the dehydrocholic acid of equivalent melting point. The coloured juice is lastly precipitated by dilute acetic acid and is crystallized in alcohol at 80 C. then treated by the same process.

Several similar operations permit 351 gr. of dehydrocholic acid (melting point 238-239 C.) to be obtained constituting a total yield of 70%.

The invention is not limited to the described modes of carrying out the invention which were given by way of example. For instance, the aforementioned aliphatic hydrohalogenated carbons may be replaced by others without affecting the result.

Having now described our invention what we claim asnew and desire to Asecure by Letters Patent is:

1. Process of purifying dehydrocholic acid that comprises the steps of treating said dehydrocholic acid in the impure state with an aqueous alkaline solution wherein it is dissolved in the form of its alkaline salt, thereafter adding to halogenated hydrocarbon-dehydrocholic this solution at least one halogenated aliphatic hydrocarbon thereby precipitating therefrom an addition compound of halogenated hydrocarbondehydrocholic acid, thereafter separating this precipitate, thereafter decomposing it by excess hot water thereby regenerating the alkaline salt of said acid, thereafter liberating this dehydro` cholic acid in the pure state by acidification.

2. Process of purifying dehydrocholic acid as claimed in claim 1, in which the concentration of said dehydrocholic acid with respect to said gkaline solution is comprised between 20 and 3. Process of purifying dehydrocholic acid as claimed in claim 1, in which said halogenated hydrocarbon is utilized in a proportion at least molecularly equivalent to said dehydrocholic acid.

4. Process of purifying dehydrocholic acid as claimed in claim l, in which said halogenated hydrocarbon is chloroform. Y

5. Process of purifying dehydrocholic acid as claimed in claim 1, in which said halogenated hydrocarbon is dichlorethane.

6. Process of purifying dehydrocholic acid as claimed in claim 1, in which said halogenated hydrocarbon is carbon tetrachloride.

'7. Process of purifying dehydrocholic acid as claimed in claim 1, in which said halogenated hydrocarbon is trichlorethylene.

8. Process of purifying dehydrocholic acid as claimed in claim 1, in which said halogenated hydrocarbon is ethyl bromide.

9. Process of purifying dehydrocholic acid that comprises the steps of treating said dehydrocholic acid in the impure state with an aqueous alkaline solution, wherein it is dissolved in the form of its alkaline salt, said acid being at a concentration comprised between 2'0 and 25% with respect to said alkaline solution, thereafter adding to this solution at least one halogenated aliphatic hydrocarbon in a proportion at least molecularly equivalent to said dehydrocholic acid, thereby precipitating therefrom an addition compound of acid, thereafter separating this precipitate, thereafter decomposing it by an excess of hot water thereby regenerating the alkaline salt of said dehydrocholic acid, and thereafter` liberating this dehydrocholic acid in the pure state by acidification.

PIERRE POULENC.

BERNARD GAUTHIER.

GEORGES MARIE EMILE ALOYS KIRCHHOFFER.

No references cited. 

1. PROCESS OF PURIFYING DEHYDROCHLOLIC ACID THAT COMPRISES THE STEPS OF TREATING SAID DEHYDROCHOLIC ACID IN THE IMPURE STATE WITH AN AQUEOUS ALKALINE SOLUTION WHEREIN IT IS DISSOLVED IN THE FORM OF ITS ALKALINE SALT, THEREAFTER ADDING THIS SOLUTION AT LEAST ONE HALOGENATED ALIPHATIC HYDROCARBON THEREBY PRECIPITATING THEREFROM AN ADDITION COMPOUND OF HALOGENATED HYDROCARBONDEHYDROCHOLIC ACID, THEREAFTER SEPARATING THIS PRECIPITATE, THEREAFTER DECOMPOSING IT BY EXCESS HOT WATER THEREBY REGENERATING THE ALKALINE SALT OF SAID ACID, THEREAFTER LIBERATING THIS DEHYDROCHOLIC ACID IN THE PURE STATE BY ACIDIFICATION. 